The present invention relates to chemical modification of biomedical materials, such as collagen, chitosan, and hemoglobin, with a naturally occurring crosslinking reagent, genipin, and to biocompatible materials, useful in biological implants, adhesives, wound dressings, and blood substitutes, which are crosslinked or polymerized with genipin.
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Crosslinking of biological molecules is often desired for optimum effectiveness in biomedical applications. For example, collagen, which constitutes the structural framework of biological tissues, has been extensively used for manufacturing bioprostheses and other implanted structures, such as vascular grafts, wherein it provides a good medium for cell infiltration and proliferation. Collagen sheets are also used as wound dressings, providing the advantages of high permeability to water vapor and rapid wound healing. Disadvantages of uncrosslinked collagen include low tensile strength and ready degradation by collagenase. The fixation, or crosslinking, of collagenous tissues increases mechanical strength, reduces cleavage by collagenase, and reduces antigenicity and immunogenicity.
Chitosan, a deacetylated derivative of chitin, contains free amino groups which may also be crosslinked, e.g. by glutaraldehyde (Jameela), and has been used or proposed for use in implanted drug-delivery devices, skin substitutes, wound dressings, and other biomaterials. It has shown the beneficial property of reducing calcification when coated on other implanted materials (Chanda).
Various crosslinking reagents have been used in the chemical modification of amine-containing biomedical materials. The most common are synthetic chemicals such as formaldehyde, glutaraldehyde, dialdehyde starch, glycoaldehyde, cyanamide, diimides, and diisocyanates. Of these, glutaraldehyde, which reacts rapidly with proteins, is the most commonly used.
Problems which have been encountered with crosslinked collagen include the tendency to calcify when implanted, leading to stiffness around an implant, eventual degradation and resorption into the surrounding tissue, and toxic reactions to crosslinking reagents. Glutaraldehyde is known to have allergenic properties, causing occupational dermatitis, and is cytotoxic at concentrations greater than 10-25 ppm and as low as 3 ppm in tissue culture.
Another useful application of crosslinked biological materials is in blood substitutes. Cell-free hemoglobin, a blood substitute, is useful for antigen-free blood transfusions, but is easily transformed from tetramer to dimer during circulation. High oxygen affinity and short half-life are additional limitations of this material. These difficulties may be overcome by chemical modification of hemoglobin with a crosslinking reagent (see e.g. Chang, Keipert). The increase of the half-life of hemoglobin in circulation and the reduction of its oxygen affinity can be achieved by both intermolecular and intramolecular crosslinking. Crosslinking with glutaraldehyde, however, has been found to induce dimer formation during hemoglobin polymerization.
It is therefore desirable to provide a crosslinking reagent suitable for use in biomedical applications which is of low toxicity, forms stable, biocompatible crosslinked products, and retains its stability upon implantation.
In one aspect, the invention provides a biocompatible implant or wound dressing. The implant or dressing is of a material which comprises a biocompatible crosslinked amine-containing biomolecule, selected from chitosan or a connective-tissue protein, where the biomolecule is crosslinked with genipin. Where the biomolecule is a connective-tissue protein, the protein is preferably collagen. Also provided is a biocompatible adhesive, which comprises a biocompatible crosslinked gelatin, derived from a collagenous source, where the gelatin is crosslinked with genipin.
In another aspect, the invention provides a method of manufacturing a biocompatible implant or wound dressing. The method includes the steps of fabricating a material which comprises an amine-containing biomolecule, selected from chitosan or a connective-tissue protein, into a structure suitable for the implant or dressing, and crosslinking the material with genipin. When the material comprises a connective-tissue protein, the protein is preferably collagen.
In other aspects, the invention provides a composition suitable for use in a blood substitute, which comprises hemoglobin crosslinked with genipin, and a method of preparing the composition, by treating hemoglobin with an amount of genipin effective to crosslink the hemoglobin. Preferably, the crosslinked hemoglobin has a mean degree of polymerization greater than 1 and less than about 9.